Many important biological substances, including phospholipids, oligonucleotides, deoxynucleosides, nucleotides and nucleosides, exist as symmetrical and unsymmetrical phosphodiesters. The usefulness of such phosphodiester compounds in medical applications is well known. See, e.g., Desseaux et al., "Synthesis of Phosphodiester and Triester Derivatives of AZT with Tethered N-Methyl Piperazine and N,N,N'trimethylethylenediamine," Bioorg. & Med. Chem. Letters, vol. 3, no. 8, pp. 1547-50 (1993); PCT publication no. WO 96/27379. Recently, PCT publication no. WO 96/23526, incorporated herein by reference, describes phosphodiester compounds which are useful as contrast agents for diagnostic imaging.
A number of methods of making phosphodiester compounds, based on P(III) chemistry, are known. In general, phosphorylation plays an important role in the synthesis of phosphodiester compounds. But the known phosphodiester synthetic methods all suffer from a number of problems including how phosphorylation is accomplished.
One method for making phosphodiesters involves the use of phosphoramidite chemistry. See, e.g., Bannwarth et al., "A Simple and Effective Chemical Phosphorylation Procedure for Biomolecules," Helvetica Chimica Acta, vol. 70, pp. 175-186 (1987); Bannwarth et al., "Bis(allyloxy)(diisopropylamino) phosphine as a New Phosphinylation Reagant of the Phosphorylation of Hydroxy Functions," Tetrahedron Letters, vol. 30, no. 32, pp. 4219-22 (1989); Moore et al., "Conceptual Basis of the Selective Activation of Bis(dialkylamino) methoxyphosphines by Weak Acids and Its Application toward the Preparation of Deoxynucleoside Phosphoramidites in Situ," J.Org.Chem., vol. 50, pp. 2019-2025 (1985); Hebert et al., "A New Reagant for the Removal of the 4-Methoxybenzyl Ether: Application to the Synthesis of Unusual Macrocyclic and Bolaform Phosphatidycholines," J.Org.Chem., vol. 57, pp. 1777-83 (1992); Desseaux et al., "Synthesis of Phosphodiester and Triester Derivatives of AZT with Tethered N-Methyl Piperazine and N,N,N'trimethylethylenediamine," Bioorg. & Med. Chem. Letters, vol. 3, no. 8, pp. 1547-50 (1993); Pirrung et al., "Inverse Phosphotriester DNA Synthesis Using Photochemically-Removable Dimethoxybenzoin Phosphate Protecting Groups," J.Org.Chem., vol. 61, pp. 2129-36 (1996).
Such phosphoramidite methods, however, suffer from the fact that the phosphoramidites are typically unstable compounds (both chemically and kinetically) and upon purification by distillation may ignite or cause an explosion. Further, phosphoramidite methods are generally not suitable for manufacturing phosphodiester compounds on a commercial basis. This is so because the phosphoramidite starting materials are very expensive and are not readily available, and because methods using phosphoramidites tend to involve additional process steps (e.g., additional step of cleaving protecting groups after phosphorylation) as well as multiple isolation and/or purification steps of the intermediates.
Methods involving the use of phosphodichloridates as the phosphorylating agent suffer from similar problems. See, e.g., Martin et al., "General Method for the Synthesis of Phospholipid Derivatives of 1,2-O-Diacyl-sn-glycerols," J.Org.Chem., vol. 59, pp. 4805-20 (1994); Martin et al., "A General Protocol for the Preparation of Phospholipids via Phosphate Coupling," Tetrahedron Letters, vol. 29, no. 30, pp. 3631-34 (1988); Lammers et al., "Synthesis of Phospholipids via Phosphotriester Intermediates," J.Roya Netherlands Chem. Soc'y, 98/4, pp. 243-250 (April 1979); Martin et al., "Synthesis and Kinetic Evaluation of Inhibitors of the Phosphatidylinositol-Specific Phospholipase C from Bacillus cereus," J.Org.Chem., vol. 61, pp. 8016-23 (1996).
Another method used for making phosphodiester compounds involves the use of PCl.sub.3 to generate hydrogen-phosphonate intermediates. See, e.g., Lindh et al., "A General Method for the Synthesis of Glycerophospholipids and Their Analogues via H-Phosphonate Intermediates," J.Org.Chem., vol. 54, pp. 1338-42 (1989); Garcia et al., "Synthesis of New Ether Glycerophospholipids Structurally Related to Modulator," Tetrahedron, vol. 47, no. 48, pp. 10023-34 (1991); Garigapati et al., "Synthesis of Short Chain Phosphatidylinositols," Tetrahedron Letters, vol. 34, no. 5, pp. 769-72 (1993). This method, however, requires the use of a coupling reagent which can either be purchased or independently synthesized, and thus renders such methods expensive or more complex. In addition, multiple isolation and purification steps of the intermediates are required, often with laborious drying conditions for the H-phosphonate intermediate.
Consequently, there remains a need for a safe, efficient and inexpensive process for the production, in high yields, of phosphodiester compounds with the potential of having a wide variety of substituents which does not require either the use of a protecting group or a coupling agent. In particular, there remains a need for a process which could be performed in one reaction vessel and does not require multiple isolation and purification steps because of the formation of multiple intermediates.